The ΔKILR motif is required for Cdc20 to bind and activate the APC/C. (A) The ΔKILR mutant cannot activate the APC/C in vitro. Wild-type, RA, or ΔKILR mutants of Cdc20 were purified from baculovirus-infected insect cells, and their ability to activate the APC/C was assayed using securin as a substrate. The APC/C was prepared from mitotic cells depleted of Cdc20. Results are representative of two experiments. (B and C) The ΔKILR mutant is defective in binding to the APC/C. (B) HeLa cell lines expressing inducible 3×Flag–wild type, RA, or ΔKILR mutant Cdc20 were treated with siRNA against Cdc20 for 48 h. 9 h after release from a thymidine block, cells were treated with 1 µM reversine + 10 µM MG132 for 3 h and harvested by mitotic shake off. The APC/C was immunoprecipitated with anti-APC4 antibodies and analyzed by quantitative immunoblotting. (C) In vitro translated (IVT) 3×Flag–wild-type, RA, or ΔKILR mutant Cdc20 was incubated with mitotic extracts depleted of endogenous Cdc20, and the APC/C was immunoprecipitated with anti-APC4 antibodies before immunoblotting with anti-APC4 and anti-Flag epitope antibodies. Results in B and C are representative of three independent experiments. end, endogenous; IP, immunoprecipitation; Ubi, ubiquitin; WT, wild type.